Abstract
In principle, treatment of embryonic kidneys growing in organ culture with short interfering RNA (siRNA) offers a powerful means of investigating molecular function quickly and cheaply. Experiments using this approach have yielded significant new data, but they have also highlighted important limitations. Here, we briefly describe the published successes and limitations and present detailed instructions for two methods of siRNA treatment. The first method applies siRNA to intact cultured kidneys; this method is the quicker and easier of the two, but it is the one most affected by problems of siRNA uptake by certain renal tissues. The second method reduces kidney rudiments to a suspension of single cells, applies siRNA at that stage, when the cells are highly accessible, and then reaggregates the kidney; this method is more time-consuming but suffers less from problems of limited uptake. As well as proving instructions for the methods, we provide a brief discussion of necessary controls.
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References
Elbashir SM, Harborth J, Lendeckel W et al (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498
Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200
Sakai T, Larsen M, Yamada KM (2003) Fibronectin requirement in branching morphogenesis. Nature 423:876–881
Davies JA, Ladomery M, Hohenstein P et al (2004) Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation. Hum Mol Genet 13:235–246
Lee WC, Berry R, Hohenstein P, Davies J (2008) siRNA as a tool for investigating organogenesis: the pitfalls and the promises. Organogenesis 4:176–181
Lee WC, Hough MT, Liu W et al (2010) Dact2 is expressed in the developing ureteric bud/collecting duct system of the kidney and controls morphogenetic behavior of collecting duct cells. Am J Physiol Renal Physiol 299:F740–F751
Berry R, Harewood L, Pei L et al (2010) Esrrg functions in early branch generation of the ureteric bud and is essential for normal development of the renal papilla. Hum Mol Genet 20:917–926
Li X, Hyink DP, Radbill B et al (2009) Protein kinase-X interacts with Pin-1 and Polycystin-1 during mouse kidney development. Kidney Int 76:54–62
Sheng W, Wang G, La Pierre DP et al (2006) Versican mediates mesenchymal–epithelial transition. Mol Biol Cell 17:2009–2020
Kim HS, Kim MS, Hancock AL et al (2007) Identification of novel Wilms’ tumor suppressor gene target genes implicated in kidney development. J Biol Chem 282:16278–16287
Michael L, Sweeney DE, Davies JA (2007) The lectin Dolichos biflorus agglutinin is a sensitive indicator of branching morphogenetic activity in the developing mouse metanephric collecting duct system. J Anat 210:89–97
Maeshima A, Vaughn DA, Choi Y, Nigam SK (2006) Activin A is an endogenous inhibitor of ureteric bud outgrowth from the Wolffian duct. Dev Biol 295:473–485
Unbekandt M, Davies JA (2009) Dissociation of embryonic kidneys followed by reaggregation allows the formation of renal tissues. Kidney Int 77:407–416
Siegel N, Rosner M, Unbekandt M et al (2010) Contribution of human amniotic fluid stem cells to renal tissue formation depends on mTOR. Hum Mol Genet 19:3320–3331
Davies JA (2010) The embryonic kidney: isolation, organ culture, immunostaining and RNA interference. Methods Mol Biol 633:57–69
Acknowledgments
This work was supported by NC3Rs grant G0700480 and EU Star-t-Rek network FP7 223007.
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Davies, J.A., Unbekandt, M. (2012). siRNA-Mediated RNA Interference in Embryonic Kidney Organ Culture. In: Michos, O. (eds) Kidney Development. Methods in Molecular Biology™, vol 886. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-851-1_26
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DOI: https://doi.org/10.1007/978-1-61779-851-1_26
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